The long-term goal of this research is to understand the activation of oxygen and subsequent selective oxidation of hydrocarbons that occurs at non-heme diiron centers in the hydroxylase enzymes of bacterial multicomponent monooxygenases (BMMs). Studies of both native and genetically modified systems, including soluble methane monooxygenase (sMMO), toluene/o-xylene monooxygenase (ToMO), and alkene monooxygenase (AMO), will enable us to elucidate the carefully orchestrated delivery of four substrates - electrons, protons, O2, and a hydrocarbon (RH) - to their diiron centers to generate product (ROH = alkyl alcohol, aryl alcohol, or epoxide) and water. Comparisons among the three enzyme systems will sharpen our understanding of features that control the nature of the oxygen activation and utilization steps. The BMMs each have several component proteins: a diiron-containing hydroxylase, an electron-donating reductase, a regulatory protein, and, in the case of ToMO, a Rieske protein. Interactions between these components will be studied to reveal features by which the monooxygenase systems control O2 activation and hydrocarbon oxidation to catalyze the selective hydroxylation or epoxidation of their respective substrates. In parallel work, synthetic model compounds will be prepared to mimic the geometric and electronic structures of the hydroxylase diiron centers. Reactions of these models with O2 and substrates will be characterized to calibrate assignments for species invoked in related enzyme chemistry. Both proteins and synthetic models will be studied by X-ray crystallography to establish geometry. Electronic, vibrational, X-ray absorption, M"ssbauer, CD/MCD, and advanced EPR techniques, some in collaboration with experts in these areas, will be applied to investigate electronic stuctures and, in pre-steady state kinetic studies, stopped-flow optical and freeze-quench methods will be used to identify and characterize transient intermediates in the enzyme reaction cycles. Extensive use of site-directed mutagenesis, especially for the toluene/o-xylene system, will enable working hypotheses about specific pathways by which the four substrates access the diiron active sites to be evaluated. The synthesis of accurate biomimetic model compounds will benefit from efficient new designs for preparing dinucleating ligands using macrocycles and dendrimer sheaths as well as from a novel strategy that employs a pendant bridging carboxylate arm. Because of their ability to degrade hydrocarbons, BMMs have been applied for the bioremediation of contaminated seawater, polluted soil environments, and impure drinking water. Their ability to selectively oxidize substrates renders them valuable for synthetic applications in the pharmaceutical and chemical industries. Knowledge of the basic mechanisms of O2 activation and hydrocarbon hydroxylation provided by this research has far-reaching consequences for related non-heme diiron enzymes and will help to establish fundamental principles relating macromolecular structure and function in many other metalloproteins.

Public Health Relevance

By studying both natural systems and synthetic models, this research will reveal how methane-utilizing and related bacteria selectively oxidize hydrocarbons for energy and food by activating oxygen at iron centers housed in their hydroxylase enzymes. The ability of these hydrocarbon-consuming "superbugs" to degrade chlorinated hydrocarbons has led to their application in the bioremediation of industrial wastewater, decontamination of seawater, and removal of soil pollutants.